CN109738405B - Method for quantitatively determining flavonoid compounds - Google Patents
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Abstract
A method for quantitatively determining flavonoid compounds belongs to the technical field of nano material preparation and chemical analysis and detection. The invention prepares a reversible nano-porphyrin fluorescence sensor with double composite nano-effect by utilizing self-assembled nano-tetra- (4-pyridyl) zinc porphyrin photosensitive effect and ZnCdSe quantum dots in a buffer solution system; and then, by utilizing the strong acting force between the flavonoid compound and the nano porphyrin, the reversible composite sensing interface between the nano porphyrin and the quantum dot is pulled apart by different degrees of specific change, so that the quantitative detection of the flavonoid compound is realized. The preparation method provided by the invention is simple and controllable, has high sensitivity, good selectivity and strong specificity to a flavonoid compound detection method, can play an important role in flavonoid compound detection, and is expected to have important application value in the fields of biochemistry and the like.
Description
Technical Field
The invention belongs to the technical field of nano material preparation and chemical analysis and detection, and particularly relates to controllable preparation of a novel reversible nano porphyrin fluorescence sensor and a method for detecting flavonoid compounds with high sensitivity by using the novel reversible nano porphyrin fluorescence sensor.
Background
The flavonoids are the products of plant secondary metabolism, widely exist in various foods and beverages such as tea, coffee, wine, fruits, vegetables and whole wheat grains, and have obvious pharmacological activity such as antioxidation, anti-inflammation, antivirus and the like. To date, a variety of analytical methods have been used to detect polyphenols, including electrochemical high performance liquid chromatography, capillary electrophoresis, electrospray mass spectrometry, high-speed counter-current chromatography, and diode arrays. Although these methods have high precision, high sensitivity and small cross interference, the experimental process requires complicated sample pretreatment, is time-consuming and expensive, and can only be operated by trained professionals, which is inconvenient for field detection. Therefore, an efficient, simple and reliable means for detecting flavonoids is needed.
Disclosure of Invention
One of the purposes of the invention is to provide a controllable preparation method of a novel reversible nano porphyrin fluorescence sensor, which has simple preparation and mild reaction conditions; the second purpose is to provide a reversible nano porphyrin fluorescence sensor which has high sensitivity and good selectivity and can quickly and quantitatively identify quercetin, rutin, kaempferide and kaempferol based on a fluorescence on-off-on mode method.
The reversible nano-porphyrin fluorescence sensor for specifically identifying and quantifying quercetin, rutin, kaempferide and kaempferol adopts ZnCdSe quantum dots as a fluorescence probe, self-assembled nano-porphyrin prepared from a tetra- (4-pyridyl) zinc porphyrin solution and betaine is a fluorescence quencher, and the two are specifically combined to obtain the switch nano-porphyrin fluorescence sensor. The reversible (on-off-on) nano porphyrin fluorescence sensor is obtained by the action of the on-off nano porphyrin fluorescence sensor and quercetin, rutin, kaempferide and kaempferol.
A method for quantitatively determining flavonoid compounds is characterized by comprising the following specific steps:
(1) dissolving zinc dichloride and N-acetyl-L-cysteine in ultrapure water, stirring for 20 minutes in an ice bath under normal pressure, adjusting the pH of the solution to 9.7 by using a sodium hydroxide solution, adding cadmium dichloride, filling nitrogen, stirring in the ice bath for 5 minutes. NaHSe was added and stirred for 5 minutes. Finally, putting the solution into a reaction kettle, and reacting for 65 minutes in a drying oven at the temperature of 200 ℃ to obtain ZnCdSe quantum dots;
(2) dissolving tetra- (4-pyridyl) zinc porphyrin, namely ZnTPyP, in N, N-Dimethylformamide (DMF), ultrasonically dissolving, and placing at 4 ℃ for later use; then adding a DMF solution of ZnTPyP and an aqueous solution of dodecyl dimethyl betaine, mixing and stirring for 10min at room temperature to obtain a very stable green transparent colloid tetra- (4-pyridyl) zinc porphyrin self-assembly nanorod, namely a nano porphyrin solution;
(3) adding a tetra- (4-pyridyl) zinc porphyrin self-assembly solution into a ZnCdSe quantum dot fluorescent probe, adding a Tris-HCl buffer solution with the pH value of 8.0, quenching the fluorescence of the quantum dot by the tetra- (4-pyridyl) zinc porphyrin self-assembly through electron transfer and fluorescence resonance energy transfer, and providing a 'Turn-off' state of the quantum dot through a compound obtained by specific combination, wherein the fluorescence intensity is reduced from about 840 to about 360; thereby obtaining the switch nano porphyrin fluorescence sensor with double composite nano effect;
(4) flavonoid compounds with different standard concentrations are respectively added into the same switch nano porphyrin fluorescence sensor, the fluorescence of quantum dots in the switch nano porphyrin fluorescence sensor is recovered, and the phenomenon that the fluorescence of the quantum dots is recovered by the flavonoid compounds with different concentrations generates obvious difference, so that the identification and the quantification of the flavonoid compounds in a reversible nano porphyrin fluorescence sensing mode are realized; thereby obtaining the reversible 'on-off-on' nano porphyrin fluorescence sensor; a standard phenomenon is obtained in that,
or directly combining the steps (3) and (4): mixing flavonoids compounds with different concentration ranges with the tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution with the pH value of 8.0, and standing for 5 minutes; then adding the ZnCdSe quantum dots synthesized in the step (1), performing fluorescence spectrum measurement at 400-550nm, and measuring the spectrum after 5 minutes to obtain a standard spectrum;
(5) adding the flavonoid compound solution with the concentration to be measured into the switched nano porphyrin fluorescence sensor with the same step (4), or operating according to the step of obtaining the standard spectrum, and contrasting the obtained phenomenon or spectrum with the step (4) to obtain the concentration;
further preferably:
the ratio of the amounts of zinc dichloride, N-acetyl-L-cysteine, cadmium dichloride and NaHSe in the invention is as follows: 1.0:3.0:0.01:0.1, and the emission wavelength of the ZnCdSe quantum dot fluorescent probe in the general step (1) is 465-480 nm;
in the step (2), the mass ratio of the tetra- (4-pyridyl) zinc porphyrin to the dodecyl dimethyl betaine is 1: 14-16;
the mass ratio of the tetra- (4-pyridyl) zinc porphyrin nanorod in the step (3) to the ZnCdSe quantum dot solution is 277-280: 1;
in the invention, the concentration of the tetra- (4-pyridyl) zinc porphyrin nanorod and the concentration of the ZnCdSe quantum dot in the mixed solution in the step (3) are respectively 0.84-5.04 mu mol/L and 4.2 multiplied by 10-9mol/L。
Further preferably: the reversible nano porphyrin fluorescence sensor is a compound obtained by the specific combination of quantum dots and nano porphyrin. The fluorescence intensity is reduced from 840 to 360.
The reversible nano porphyrin fluorescence sensor has high sensitivity. The fluorescence intensity of the ZnCdSe quantum dot fluorescent probe is gradually weakened along with the increase of the tetra- (4-pyridyl) zinc porphyrin self-assembly solution, even can be quenched to the end, and the qualitative and quantitative detection of the step (4) can be realized as long as the partial quenching or the complete quenching (preferably in the range of a linear relation part) is carried out; the concentration of the tetra- (4-pyridyl) zinc porphyrin nanorod (0.84-5.04 mu mol/L) and ZnCdSe quantum dots (4.2 multiplied by 10)-9mol/L) of the fluorescence intensity to form a good linear relation; the fluorescence intensity of quercetin, rutin, kaempferol and kaempferol is detected to have a linear relation in a certain range.
The reversible nano porphyrin fluorescence sensor quantitatively detects the concentration of quercetin from (2.0 multiplied by 10)-8～1.0×10-7mol/L). The concentration of quercetin is increased, and the linear relationship is good. The linear correlation coefficients are 0.9670 respectively. Rutin (1.0X 10)-8～1.0×10-7mol/L) and the fluorescence intensity after the reversible nano porphyrin fluorescence sensor is combined is enhanced along with the increase of the concentration of rutin and has a linear relation. The linear correlation coefficients are 0.8974 respectively. Kaempferol (5.0X 10)-9-1.0×10-8mol/L) and the fluorescence intensity after the reversible nano porphyrin fluorescence sensor is combined is enhanced along with the increase of the concentration of the kaempferol, and a good linear relation is formed. The linear correlation coefficients are 0.9940 respectively. Kaempferide (5.0X 10)-9-1.0×10-8mol/L) and the fluorescence intensity after the reversible nano porphyrin fluorescence sensor is combined is enhanced along with the increase of the concentration of the kaempferide, and a good linear relation is formed. The linear correlation coefficients are 0.9992 respectively. Thereby obtainingInverse on-off-on nano porphyrin fluorescence sensor; so that the steps (3) and (4) are combined and separated to obtain the same effect.
The reversible nano porphyrin sensor has good stability. The reversible nano porphyrin fluorescence sensor is 1.0 multiplied by 10- 5mol/L ion (KCl, Na)2SO4、CaCl2、Mg2SO4、ZnCl2) Under the condition of mixed interference of 1 mu g/mL biological matrix (cell culture fluid, calf plasma, newborn bovine serum and bovine serum albumin) and 1 mu g/mL, the intensity of fluorescence recovery of the biological matrix and the quercetin, the rutin, the kaempferol and the kaempferol is almost unchanged
The reversible nano porphyrin fluorescence sensor has high response speed to flavonoids. After quercetin, rutin, kaempferol and kaempferol are added into the reversible nano porphyrin fluorescence sensor, the fluorescence is rapidly recovered, and the most stable value is reached within 5 minutes.
Compared with the traditional method for measuring flavonoids by a chromatography method, the method has the advantages of simple preparation, mild reaction conditions, high sensitivity for detecting flavonoids, strong anti-interference capability and good response, and the nano porphyrin fluorescence sensor has practical application value in the fields of biochemistry, food, medicine and the like.
Drawings
FIG. 1 is a schematic diagram of a controllable preparation method of the novel reversible (on-off-on) nano porphyrin fluorescence sensor and a method for detecting flavonoids with high sensitivity.
FIG. 2 shows the UV-VIS spectrum of the tetra- (4-pyridyl) zinc porphyrin self-assembly solution in the reversible nano-porphyrin sensor of the invention, with the abscissa as wavelength and the ordinate as absorbance.
FIG. 3 is a transmission electron microscope photograph of a tetra- (4-pyridyl) zinc porphyrin self-assembly solution in the reversible nano-porphyrin sensor of the present invention, which is a nanorod.
FIG. 4 is a fluorescence spectrum of a reversible nano-porphyrin sensor after specific binding of ZnCdSe quantum dots and tetra- (4-pyridyl) zinc porphyrin self-assembly solution, with the abscissa as wavelength and the ordinate as fluorescence intensity.
FIG. 5 is a graph showing the sensitivity of the reversible nano-porphyrin sensor of the present invention. And (3) a fluorescence spectrogram of the tetra- (4-pyridyl) zinc porphyrin self-assembly solution (0.84-5.04 mu mol/L) and ZnCdSe quantum dots after the action of a Tris-HCl buffer solution (pH 8.0), wherein the abscissa is the wavelength and the ordinate is the fluorescence intensity.
FIG. 6 shows reversible nano-porphyrin sensor and quercetin (2.0 × 10) with different concentrations-8～1.0×10-7mol/L) of the fluorescence recovery spectrum after the action, the abscissa is the wavelength, and the ordinate is the fluorescence intensity.
FIG. 7 shows reversible nano-porphyrin sensor and rutin (1.0 × 10) with different concentrations-8～1.0×10-7mol/L) of the fluorescence recovery spectrum after the action, the abscissa is the wavelength, and the ordinate is the fluorescence intensity.
FIG. 8 shows reversible nano-porphyrin sensor and kaempferol (5.0X 10) with different concentrations according to the present invention-9-1.0×10-8mol/L) of the fluorescence recovery spectrum after the action, the abscissa is the wavelength, and the ordinate is the fluorescence intensity.
FIG. 9 shows reversible nano-porphyrin sensor and kaempferide (5.0 × 10) with different concentrations according to the present invention-9-1.0×10-8mol/L) of the fluorescence recovery spectrum after the action, the abscissa is the wavelength, and the ordinate is the fluorescence intensity.
FIG. 10 is a linear correlation diagram of the reversible nano-porphyrin sensor of the present invention after the interaction with quercetin of different concentrations, the abscissa is the concentration of quercetin, and the ordinate is the fluorescence recovery intensity (F)2) And original fluorescence intensity (F) of ZnCdSe quantum dot0) The ratio of (a) to (b).
FIG. 11 is a linear correlation diagram of the reversible nano-porphyrin sensor of the invention after the interaction with rutin of different concentrations, with the abscissa being the concentration of rutin and the ordinate being the ratio of the fluorescence recovery intensity to the original fluorescence intensity of ZnCdSe quantum dots.
FIG. 12 is a linear correlation graph of the reversible nano-porphyrin sensor of the invention after the action with kaempferol of different concentrations, with the abscissa being the concentration of kaempferol and the ordinate being the fluorescence recovery intensity (F)2) And original fluorescence intensity (F) of ZnCdSe quantum dot0) The ratio of (a) to (b).
FIG. 13 is a linear correlation diagram of the reversible nano-porphyrin sensor of the invention after the action with kaempferide of different concentrations, with the abscissa being the concentration of kaempferide and the ordinate being the ratio of the fluorescence recovery intensity to the original fluorescence intensity of ZnCdSe quantum dots.
FIG. 14 shows the stability of the reversible nano-porphyrin sensor of the present invention. Reversible nano-porphyrin sensor and quercetin in Ca2+、Zn2+、Mg2+、K+、Na+Cell culture fluid, calf plasma, newborn bovine serum, bovine serum albumin and mixed interference (mix) under the condition of post-action stability. The abscissa is the added interfering substance and the ordinate is the fluorescence recovery intensity (F)2) And original fluorescence intensity (F) of ZnCdSe quantum dot0) The ratio of (a) to (b).
FIG. 15 shows the stability of the reversible nano-porphyrin sensor of the present invention. Reversible nano porphyrin sensor and rutin in Ca2+、Zn2+、Mg2+、K+、Na+Cell culture fluid, calf plasma, newborn bovine serum, bovine serum albumin and mixed interference (mix) under the condition of post-action stability. The abscissa is the added interfering substance and the ordinate is the fluorescence recovery intensity (F)2) And original fluorescence intensity (F) of ZnCdSe quantum dot0) The ratio of (a) to (b).
FIG. 16 is a graph showing the stability of the reversible nano-porphyrin sensor of the present invention. Reversible nano porphyrin sensor and kaempferol in Ca2+、Zn2+、Mg2+、K+、Na+Cell culture fluid, calf plasma, newborn bovine serum, bovine serum albumin and mixed interference (mix) under the condition of post-action stability. The abscissa is the added interfering substance and the ordinate is the fluorescence recovery intensity (F)2) And original fluorescence intensity (F) of ZnCdSe quantum dot0) The ratio of (a) to (b).
FIG. 17 shows the stability of the reversible nano-porphyrin sensor of the present invention. Reversible nano porphyrin sensor and kaempferol in Ca2+、Zn2+、Mg2+、K+、Na+Cell culture fluid, calf plasma, newborn bovine serum, bovine serum albumin and mixed interference (mix) under the condition of post-action stability. Abscissa of the circleThe ordinate represents the fluorescence recovery intensity (F) for the added interfering substances2) And original fluorescence intensity (F) of ZnCdSe quantum dot0) The ratio of (a) to (b).
Detailed Description
The present invention will be described in further detail with reference to specific examples below so that those skilled in the art can more clearly understand the present invention. The following should not be construed as limiting the scope of the claimed invention.
Example (b):
the chemicals and solvents used in the examples were all analytical grade. The experimental operation is a magnetic stirring mode. The fluorescence spectrum determination conditions are all emission wavelength of 400-550nm, excitation wavelength of 360nm and slit width of 10-15 nm.
Example 1: the identification and quantitative analysis of the reversible nano porphyrin fluorescence sensor to the quercetin are shown in the schematic diagram of the method as 1, and the steps are as follows:
(1) synthesis of ZnCdSe quantum dot fluorescent probe
Zinc dichloride (0.035g,6.4mM) and N-acetyl-L-cysteine (0.1253g,19.2mM) were dissolved in 40mL of ultrapure water, stirred in an ice bath at normal pressure for 20 minutes, then the solution was adjusted to pH 9.7 with sodium hydroxide solution, 100. mu.L of cadmium dichloride (0.00058g,0.237mM) was added to the adjusted pH, and then stirred in a nitrogen-filled ice bath for 5 to 10 minutes. NaHSe was added and stirred for 5 minutes. Finally, the solution is put into a reaction kettle and reacted for 65 minutes in an oven at 200 ℃. Cooled to room temperature to give 4.9X 10-9And (3) a mol/L ZnCdSe quantum dot fluorescent probe.
(2) Synthesis of nanoporphyrin solutions
Dissolving appropriate amount of tetra- (4-pyridyl) zinc porphyrin in N, N-dimethylformamide solution to obtain a solution with a concentration of 3 × 10- 4The ultraviolet spectrum of the solution of the tetra- (4-pyridyl) zinc porphyrin N, N-dimethylformamide in mol/L is shown in figure 2. A round bottom flask was charged with 2.8ml of ZnTPyP in DMF and 45.5ml of 2.7% dodecyl dimethyl betaine and stirred at room temperature for 10min to give a very stable green transparent colloidal solution. The porphyrin nanocrystallization is observed by an ultraviolet-visible spectrophotometer to obtain the nanoThe concentration of the Michiorinated porphyrin is 1.68 multiplied by 10-5The ultraviolet spectrum of the product is shown in figure 2. The transmission electron microscope characterization shows that the nano-rods have the particle size of about 160nm, as shown in FIG. 3.
(3) Preparation of switch nano porphyrin fluorescence sensor
Add 80. mu.L of 4.9X 10 to a 1.5mL cuvette-9And (2) performing fluorescence spectrum measurement at 400-550nm by using mol/L of the ZnCdSe quantum dots synthesized in the step (1) and a Tris-HCl buffer solution of 890 mu LpH-8.0, and obtaining a peak with the fluorescence intensity of 840 at 476nm, as shown in a graph 4.
Add 80. mu.L of 4.9X 10 to a 1.5mL cuvette-9mol/L ZnCdSe quantum dots synthesized in the step (1) and 300 mu L of 1.68 multiplied by 10-5mol/L of the tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2), adding 620 mu LpH-8.0 Tris-HCl buffer solution, mixing for 5 minutes, performing fluorescence spectrum measurement at 400-550nm, and obtaining a peak with the fluorescence intensity of 350 at 474nm, as shown in FIG. 4.
(4) Quantitative analysis of quercetin by reversible nano porphyrin fluorescence sensor
Adding 100 μ L quercetin water solution into 1.5mL cuvette, 300 μ L1.68 × 10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9And (2) performing fluorescence spectrum measurement on the ZnCdSe quantum dots synthesized in the mol/L step (1) at the position of 400-550nm, and measuring the spectrum after 5 minutes. Quercetin (2.0 × 10)-8～1.0×10-7mol/L) fluorescence intensity after binding to the nano-porphyrin fluorescence sensor increased with increasing concentration of quercetin as shown in FIG. 6, and the linear correlation coefficient was 0.9670 as shown in FIG. 10.
Add 100. mu.L Quercetin (2.0X 10) to a 1.5mL cuvette-8mol/L), 100. mu.L of interfering substance (1.0X 10)- 6mol/L)、300μL1.68×10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9The ZnCdSe quantum dots synthesized in the mol/L step (1) are measured by a fluorescence spectrum at the position of 400-550nm, and the spectrum and the fluorescence after 5 minutes are measuredThe optical recovery is almost free from the influence of interference factors, and shows strong anti-interference capability, as shown in fig. 14.
Example 2: the reversible nano porphyrin fluorescence sensor quantitatively analyzes rutin, the schematic diagram of the method is shown as 1, and the steps are as follows:
(1) synthesis of ZnCdSe quantum dot fluorescent probe
The method of the step (1) in the example 1 is adopted to synthesize the ZnCdSe quantum dot fluorescent probe.
(2) Synthesis of tetra- (4-pyridyl) zinc porphyrin self-assembly solution
A tetra- (4-pyridyl) zinc porphyrin self-assembly solution was synthesized by the method of the step (2) in example 1.
(3) Preparation of switch nano porphyrin fluorescence sensor
The method of step (3) in example 1 was used to prepare a nano-porphyrin fluorescence sensor.
(4) Quantitative analysis of reversible nano porphyrin fluorescence sensor to rutin
Adding 100 μ L rutin water solution into 1.5mL cuvette, 300 μ L1.68 × 10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9And (2) performing fluorescence spectrum measurement on the ZnCdSe quantum dots synthesized in the mol/L step (1) at the position of 400-550nm, and measuring the spectrum after 5 minutes. Rutin (1.0X 10)-8～1.0×10-7mol/L) of the fluorescence intensity after being combined with the nano porphyrin fluorescence sensor is enhanced along with the increase of the concentration of rutin, as shown in figure 7, and the linear correlation coefficient is 0.8974, as shown in figure 11. Adding 100 μ L rutin (1.0 × 10) into 1.5mL cuvette-8mol/L), 100. mu.L of interfering substance (1.0X 10)-5mol/L)、300μL1.68×10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9The ZnCdSe quantum dots synthesized in the mol/L step (1) are measured by a fluorescence spectrum at the position of 400-550nm, and the spectrum after 5 minutes is measured, so that the fluorescence recovery is hardly influenced by interference factors and shows strong anti-interference capability, as shown in figure 15.
Example 3: the reversible nano porphyrin fluorescence sensor is used for quantitatively analyzing kaempferol, the schematic diagram of the method is shown as 1, and the steps are as follows:
(1) synthesis of ZnCdSe quantum dot fluorescent probe
The method of the step (1) in the example 1 is adopted to synthesize the ZnCdSe quantum dot fluorescent probe.
(2) Synthesis of tetra- (4-pyridyl) zinc porphyrin self-assembly solution
A tetra- (4-pyridyl) zinc porphyrin self-assembly solution was synthesized by the method of the step (2) in example 1.
(3) Preparation of switch nano porphyrin fluorescence sensor
The method of step (3) in example 1 was used to prepare a nano-porphyrin fluorescence sensor.
(4) Quantitative analysis of kaempferol by reversible nano porphyrin fluorescence sensor
To a 1.5mL cuvette was added 100. mu.L of an aqueous solution of kaempferol, 300. mu.L of 1.68X 10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9And (2) performing fluorescence spectrum measurement on the ZnCdSe quantum dots synthesized in the mol/L step (1) at the position of 400-550nm, and measuring the spectrum after 5 minutes. Kaempferol (5.0X 10)-9-1.0×10-8mol/L) of the fluorescence intensity after being combined with the nano porphyrin fluorescence sensor is increased along with the increase of the concentration of the kaempferol, as shown in FIG. 8, and the linear correlation coefficient is 0.9940, as shown in FIG. 12. Add 100. mu.L of kaempferol (5.0X 10) to a 1.5mL cuvette-9mol/L), 100. mu.L of interfering substance (1.0X 10)-5mol/L)、300μL1.68×10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9The ZnCdSe quantum dots synthesized in the mol/L step (1) are measured by a fluorescence spectrum at the position of 400-550nm, and the spectrum after 5 minutes is measured, so that the fluorescence recovery is hardly influenced by interference factors and shows strong anti-interference capability, as shown in figure 16.
Example 4: the reversible nano porphyrin fluorescence sensor is used for quantitatively analyzing kaempferol, the schematic diagram of the method is shown as 1, and the steps are as follows:
(1) synthesis of ZnCdSe quantum dot fluorescent probe
The method of the step (1) in the example 1 is adopted to synthesize the ZnCdSe quantum dot fluorescent probe.
(2) Synthesis of tetra- (4-pyridyl) zinc porphyrin self-assembly solution
A tetra- (4-pyridyl) zinc porphyrin self-assembly solution was synthesized by the method of the step (2) in example 1.
(3) Preparation of switch nano porphyrin fluorescence sensor
The method of step (3) in example 1 was used to prepare a nano-porphyrin fluorescence sensor.
(4) Quantitative analysis of kaempferol by reversible nano porphyrin fluorescence sensor
To a 1.5mL cuvette was added 100. mu.L of kaempferide, 300. mu.L of 1.68X 10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9And (2) performing fluorescence spectrum measurement on the ZnCdSe quantum dots synthesized in the mol/L step (1) at the position of 400-550nm, and measuring the spectrum after 5 minutes. Kaempferide (5.0X 10)-9-1.0×10-8mol/L) of the fluorescence intensity after binding with the nano porphyrin fluorescence sensor increases with the increasing concentration of kaempferide, as shown in FIG. 9, and the linear correlation coefficient is 0.9992, as shown in FIG. 13. Add 100. mu.L of kaempferol (5.0X 10) to a 1.5mL cuvette-9mol/L), 100. mu.L of interfering substance (1.0X 10)-5mol/L)、300μL1.68×10-5The tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution of 520 μ LpH ═ 8.0 were allowed to stand at mol/L for 5 minutes. 80. mu.L of 4.9X 10 was added-9The ZnCdSe quantum dots synthesized in the mol/L step (1) are measured by a fluorescence spectrum at the position of 400-550nm, and the spectrum after 5 minutes is measured, so that the fluorescence recovery is hardly influenced by interference factors and shows strong anti-interference capability, as shown in FIG. 17.
Claims (8)
1. A method for quantitatively determining flavonoid compounds is characterized by comprising the following specific steps:
dissolving zinc dichloride and N-acetyl-L-cysteine in ultrapure water, stirring for 20 minutes in an ice bath under normal pressure, adjusting the pH of the solution to 9.7 by using a sodium hydroxide solution, adding cadmium dichloride, filling nitrogen, stirring in the ice bath for 5 minutes; adding NaHSe, and stirring for 5 minutes; finally, putting the solution into a reaction kettle, and reacting for 65 minutes in a drying oven at the temperature of 200 ℃ to obtain ZnCdSe quantum dots;
(2) dissolving tetra- (4-pyridyl) zinc porphyrin, namely ZnTPyP, in N, N-Dimethylformamide (DMF), ultrasonically dissolving, and placing at 4 ℃ for later use; then adding a DMF solution of ZnTPyP and an aqueous solution of dodecyl dimethyl betaine, mixing and stirring for 10min at room temperature to obtain a very stable green transparent colloid tetra- (4-pyridyl) zinc porphyrin self-assembly nanorod solution;
(3) adding a ZnCdSe quantum dot fluorescent probe into a tetra- (4-pyridyl) zinc porphyrin self-assembly nanorod solution, adding a Tris-HCl buffer solution with the pH =8.0, quenching the fluorescence of quantum dots by the tetra- (4-pyridyl) zinc porphyrin self-assembly nanorod through electron transfer and fluorescence resonance energy transfer, and providing a 'Turn-off' state of the quantum dots through a compound obtained through specific binding, wherein the fluorescence intensity is reduced from about 840 to about 360; thereby obtaining the switch nano porphyrin fluorescence sensor with double composite nano effect;
(4) flavonoid compounds with different standard concentrations are respectively added into the same switch nano porphyrin fluorescence sensor, the fluorescence of quantum dots in the switch nano porphyrin fluorescence sensor is recovered, and the phenomenon that the fluorescence of the quantum dots is recovered by the flavonoid compounds with different concentrations generates obvious difference, so that the identification and the quantification of the flavonoid compounds in a reversible nano porphyrin fluorescence sensing mode are realized; thereby obtaining a reversible 'on-off-on' nano porphyrin fluorescence sensor and obtaining a standard phenomenon;
or directly combining the steps (3) and (4): mixing flavonoids with different concentrations with the tetra- (4-pyridyl) zinc porphyrin self-assembly nanorod solution synthesized in the step (2) and a Tris-HCl buffer solution with the pH value of 8.0, and standing for 5 minutes; then adding the ZnCdSe quantum dots synthesized in the step (1), performing fluorescence spectrum measurement at 400-550nm, and measuring the spectrum after 5 minutes to obtain a standard spectrum;
the flavonoids with different concentrations refer to: quercetin concentration 2.0 × 10-8~1.0×10-7mol/L, rutin concentration 1.0X 10-8~1.0×10-7mol/L), kaempferol concentration of 5.0X 10-9-1.0×10-8mol/L, kaempferide concentration 5.0 × 10-9-1.0×10-8mol/L；
(5) And (3) adding the flavonoid compound solution with the concentration to be measured into the switched nano porphyrin fluorescence sensor with the same step (4), or operating according to the step of obtaining the standard spectrum, and contrasting the obtained phenomenon or spectrum with the step (4) to obtain the concentration.
2. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: the ratio of the amounts of zinc dichloride, N-acetyl-L-cysteine, cadmium dichloride and NaHSe is as follows: 1.0:3.0:0.01:0.1.
3. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: the emission wavelength of the ZnCdSe quantum dot fluorescent probe in the step (1) is 465-480 nm.
4. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: the mass ratio of the tetra- (4-pyridyl) zinc porphyrin to the dodecyl dimethyl betaine in the step (2) is 1: 14-16.
5. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: and (3) the mass ratio of the tetra- (4-pyridyl) zinc porphyrin self-assembly nanorod to the ZnCdSe quantum dot is 277-280: 1.
6. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: step (3) finally, the tetra- (4-pyridyl) zinc porphyrin self-assembly nano-particles in the mixed solutionThe rod concentration and the ZnCdSe quantum dot concentration are respectively 0.84-5.04 mu mol/L and 4.2 multiplied by 10-9mol/L。
7. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: the flavonoid is selected from quercetin, rutin, kaempferol, and kaempferide.
8. The method for quantitatively determining flavonoid compounds according to claim 1, wherein: used for detecting the concentration of flavonoid compounds in ions, biological matrixes and mixed solution.
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